A Metabolic Intravascular Platform to Study FDG Uptake in Vascular Injury.

PURPOSE: Metabolic alterations underlie many pathophysiological conditions, and their understanding is critical for the development of novel therapies. Although the assessment of metabolic changes in vivo has been historically challenging, recent developments in molecular imaging have allowed us to study novel metabolic research concepts directly in the living subject, bringing us closer to patients. However, in many instances, there is need for sensors that are in close proximity to the organ under investigation, for example to study vascular metabolism. METHODS: In this study, we developed and validated a metabolic detection platform directly in the living subject under an inflammatory condition. The signal collected by a scintillating fiber is amplified using a photomultiplier tube and decodified by an in-house tunable analysis platform. For in vivo testing, we based our experiments on the metabolic characteristics of macrophages, cells closely linked to inflammation and avid for glucose and its analog 18F-fluorodeoxyglucose (18F-FDG). The sensor was validated in New Zealand rabbits, in which inflammation was induced by either a) high cholesterol (HC) diet for 16 weeks or b) vascular balloon endothelial denudation followed by HC diet. RESULTS: There was no difference in weight, hemodynamics, blood pressure, or heart rate between the groups. Vascular inflammation was detected by the metabolic sensor (Inflammation: 0.60 ± 0.03 AU vs. control: 0.48 ± 0.03 AU, p = 0.01), even though no significant inflammation/atherosclerosis was detected by intravascular ultrasound, underscoring the high sensitivity of the system. These findings were confirmed by the presence of macrophages on ex vivo aortic tissue staining. CONCLUSION: In this study, we validated a tunable very sensitive metabolic sensor platform that can be used for the detection of vascular metabolism, such as inflammation. This sensor can be used not only for the detection of macrophage activity but, with alternative probes, it could allow the detection of other pathophysiological processes.

Hypercholesterolemia impairs myocardial perfusion and permeability: role of oxidative stress and endogenous scavenging activity.

OBJECTIVES: We intended to study the effect of hypercholesterolemia (HC) on myocardial perfusion and permeability response to increased cardiac demand. BACKGROUND: Hypercholesterolemia is associated with increased incidence of cardiac events and characterized by impaired coronary vascular function, possibly mediated partly through increased pro-oxidative conditions in plasma and tissue. However, it is yet unclear whether HC is also associated with impaired myocardial perfusion and vascular permeability responses in vivo. METHODS: For 12 weeks pigs were fed a normal, HC or HC diet supplemented daily with antioxidants (HC + AO, 100 IU/kg vitamin E and 1 g vitamin C). Myocardial perfusion and vascular permeability were measured in vivo using electron beam computed tomography before and after cardiac challenge with intravenous adenosine. Plasma and tissue oxidative status was determined ex vivo. RESULTS: Plasma cholesterol increased in all cholesterol-fed pigs but was associated with increased markers of oxidative stress only in HC pigs. Myocardial perfusion increased in response to adenosine in normal and HC + AO (+37 +/- 13% and +58 +/- 22%, respectively, p < 0.05 vs. baseline) but not in HC, whereas vascular permeability index increased only in HC pigs (+ 92 +/- 25%, p = 0.002). In HC animals, tissue endogenous oxygen radical scavengers and antioxidant vitamins were depleted and LDL oxidizability enhanced, but both were normalized in HC + AO pigs. Myocardial perfusion response was directly, and permeability inversely, associated with plasma and tissue vitamin concentrations. CONCLUSIONS: This study demonstrates that experimental HC is associated with blunted myocardial perfusion and increased vascular permeability responses in vivo to increased cardiac demand, which may be partly mediated by a shift in oxidative status.

A unique myosin isoform transition in cat striated external anal sphincter muscle induced by denervation.

Denervation of limb and trunk muscles leads to characteristic changes in their biochemical properties. However, the effects of denervation of the striated external anal sphincter (EAS) muscle have not been studied, even though denervation is a common etiology for fecal incontinence. The present study reports effects ofdenervation of the cat EAS and compares them with changes in a denervated limb muscle [cat extensor digitorum longus (EDL)]. Nerves supplying the EAS and the EDL were sectioned in anesthetized cats, which were allowed to recover and live for 10, 30, or 60 days. Their muscles were then excised, and myofibrillar proteins were isolated. Myosin heavy chain (MHC) isoform composition was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The EDL and EAS showed progressive increases in MHC type IIA and progressive decreases in MHC type IIB at 10, 30, and 60 days. MHC type I progressively increased in the EDL but showed no change in the EAS. We hypothesize that this distinctive transition is related to the unique embryological origin and function of the EAS.

Human recombinant interleukin-1 beta inhibits nicotinic transmission in neurons of guinea pig pelvic plexus ganglia.

The actions of human recombinant interleukin-1 beta (hrIL-1 beta) were tested on guinea pig pelvic plexus ganglion neurons using intracellular electrophysiological methods in vitro. hrIL-1 beta caused membrane depolarization associated with a decreased input resistance or inward currents in 54% of neurons tested. hrIL-1 beta caused a hyperpolarization associated with an increase in input resistance or outward currents in 30% of neurons tested. hrIL-1 beta-evoked responses were not altered by hexamethonium (100 microM), atropine (0.5 microM), yohimbine (0.3 microM), or naloxone (1 microM), indicating that cholinergic, alpha 2-adrenergic, or opioid receptors were not involved. Drugs that inhibit Na+, Ca2+, or K+ channels did not change hrIL-1 beta-evoked responses. Stimulation of synaptic inputs to pelvic ganglion neurons evoked nicotinic cholinergic fast excitatory postsynaptic potentials (fEPSPs). hrIL-1 beta inhibited fEPSPs in 44% of neurons tested but had no effect on acetylcholine-induced depolarizations. An IL-1 beta receptor antagonist blocked all actions of hrIL-1 beta. In summary, hrIL-1 beta has excitatory and inhibitory actions on pelvic ganglion neurons. Inhibition of fEPSPs by hrIL-1 beta may be due to presynaptic inhibition of acetylcholine release.

Contractions mediated by alpha 1-adrenoceptors and P2-purinoceptors in a cat colon circular muscle.

1. The postjunctional excitatory and inhibitory effects of adrenoceptor and purinoceptor agonists and antagonists were studied in circular smooth muscle strips of cat colon. 2. In the presence of tetrodotoxin (0.5 microM), noradrenaline caused contraction or relaxation of circular smooth muscle at resting tension or with raised tone, respectively. The noradrenaline-evoked contractions were potentiated and the noradrenaline-evoked relaxations were antagonized by propranolol (1 microM), suggesting beta-adrenoceptor involvement. 3. At resting tension, noradrenaline, adrenaline and the selective alpha 1-adrenoceptor agonist, phenylephrine, caused concentration-dependent contractile responses, with EC50 values of 1.8 +/- 0.2 microM, 1.9 +/- 0.4 microM and 4.3 +/- 1.7 microM, respectively. The EC50 values and the amplitude of maximal responses were not significantly different from one another. Clonidine (0.1-500 microM), a selective alpha 2-adrenoceptor agonist, was not effective. 4. Prazosin (0.1-9 microM), competitively antagonized the contractile effects of noradrenaline with an estimated pA2 value of 6.93 and a slope of 1.07 +/- 0.03. The Kb values, estimated from a single shift (0.1 microM prazosin) of the concentration-response curves to noradrenaline, adrenaline and phenylephrine were 92.8 +/- 9.3 nM, 108.7 +/- 6.4 nM and 18.4 +/- 3.1 nM, respectively. 5. At resting tension, adenosine 5' triphosphate (ATP, 5-1000 microM), alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-MeATP, 0.05-50 microM), beta,gamma-methylene adenosine 5'-triphosphate (beta,gamma-MeATP, 0.5-100 microM), and 2-methylthioadenosine 5'-triphosphate (2-MeSATP, 1-500 microM) caused concentration-dependent contractions with EC50 values of 60.5 +/- 15.9 microM, 0.7 +/- 0.1 microM, 7.6 +/- 0.1 microM and 25.3 +/- 12.8 microM, respectively. The maximal responses to alpha, beta-MeATP and beta,gamma-MeATP were greater than maximal responses to 2-MeSATP and ATP.6. Suramin (50-500 microM), competitively antagonized the contractile responses of alpha,beta-MeATP with an estimated pA2 value of 4.92 and a slope of 1.08 +/- 0.04. The Kb values, estimated from a single shift(1I00 microM suramin) of the concentration-response curves to ATP, alpha,beta-MeATP, beta,gamma-MeATP and 2MeSATPwere 52.3 +/- 20.2 microM, 25.2 +/- 4.5 microM, 21.7 +/- 11.0 microM and 11.6 +/- 2.7 microM, respectively.7. At resting tension, reactive blue 2 (100 microM), a selective antagonist of the P2Y-purinoceptor, and 8-(p-sulphophenyl)-theophylline (8-SPT) (1 microM), a selective antagonist of the PI-purinoceptor, did not antagonize the contractile responses to alpha,beta-MeATP (0.5-5 microM). Contractile responses to ATP(50-500 microM) were not altered by 8-SPT (I microM) but were potentiated by reactive blue 2 (100 microM).8. With raised tone, ATP and 2-MeSATP caused a relaxant effect. This effect of ATP was not altered by either tetrodotoxin (TTX) (0.5 microM) or suramin (100 microM), but was antagonized by reactive blue 2(100 microM) and 8-SPT (1 microM), suggesting that inhibitory P2y- and P1-purinoceptors are involved. In contrast, alpha,beta-MeATP and Beta,gamma-MeATP caused only contractions. This contractile effect of alpha,beta-MeATPwas resistant to TTX (0.5 microM) and antagonized by suramin (100 microM).9. In summary, cat colon circular muscle contains postjunctional alpha 1-adrenoceptors and P2X purinoceptors which mediate contractions and P2y- and PI-purinoceptors which mediate relaxation.Postjunctional alpha2-adrenoceptors appear not to be present.

Effects of vasoactive intestinal contractor on voltage-activated Ca2+ currents in feline parasympathetic neurons.

Intracellular current-clamp and single-electrode voltage-clamp techniques were used to study in vitro action potentials and the action of vasoactive intestinal contractor (VIC; 0.03-1 microM) on the high-voltage-activated Ca2+ currents (ICa) of neurons in feline colonic parasympathetic ganglia. In the current-clamp recording mode, action potential amplitude was depressed by cobalt (1 mM) and omega-conotoxin (300 nM) or in nominally Ca(2+)-free Krebs solutions. In the single-electrode voltage-clamp recording mode, the ICa was isolated by blocking the voltage-gated Na+ current with tetrodotoxin (1-3 microM) and by Krebs solutions containing a low Na+ concentration. The voltage-activated K+ currents were blocked by intracellular injection of cesium through a recording electrode filled with 2 M CsCl and external application of tetraethylammonium (30-50 mM) and barium (2 mM). The Ca(2+)-dependent Cl- current was blocked by replacement of Ca2+ (2 mM) with equimolar barium. Anomalous rectification was blocked by external application of 2 mM cesium. The ICa was evoked by depolarizing step commands more positive than -40 mV from holding potentials ranging between -80 and -60 mV. ICa was depressed by cobalt (1 mM), cadmium (100 microM), and omega-conotoxin (500 nM) but not by nifedipine (10 microM), nicardipine (10 microM), and verapamil (10 microM). BAY K 8644 (3-10 microM) also did not affect the ICa. VIC (0.1-1 microM), one of the endothelin (ET) isopeptides, caused an inward current followed by an outward current. The VIC-induced inward and outward currents were associated with an increase and decrease in membrane conductance, respectively. VIC also caused an initial depression followed by a long-lasting augmentation of the ICa. ET-1, ET-2, and ET-3 equally mimicked the action of VIC on both holding current and ICa. These data suggest that VIC activates a receptor-operated channel and modulates the omega-conotoxin-sensitive voltage-activated Ca2+ channels through ETB receptor subtypes of neurons in feline colonic parasympathetic ganglia.

Stimulation of lumbar sympathetic nerves evokes contractions of cat colon circular muscle mediated by ATP and noradrenaline.

1. The action of the lumbar sympathetic nerves to cat colon was studied in vitro using isolated muscle strips with attached lumbar colonic nerves (LCN) orientated in the axis of circular muscle layer. Electrical stimulation of LCN caused frequency-dependent increases in resting tension and in amplitude of spontaneous contractions. Contractile responses were abolished by tetrodotoxin (3 microM) and by guanethidine (30 microM), indicating that they were neurogenic, involving the release of neurotransmitter from sympathetic fibres. 2. Propranolol (1-9 microM), a beta-adrenoceptor antagonist, caused a concentration-dependent potentiation of LCN-evoked contractile responses. Propranolol (3 microM) potentiated contractile responses to exogenously applied noradrenaline but not to phenylephrine. 3. Phentolamine (1-9 microM), an alpha-adrenoceptor antagonist, and prazosin (1-9 microM), an alpha 1-adrenoceptor antagonist, caused a concentration-dependent reduction of amplitude but did not abolish LCN-evoked contractile responses. Prazosin (3 microM) or phentolamine (3 microM) antagonized contractile responses to noradrenaline and phenylephrine. 4. Desensitization of purinoceptors with the P2x-receptor agonist, alpha,beta-methylene ATP, caused a decrease in amplitude of LCN-evoked contractile responses and abolished contractile responses to ATP. In muscle strips where alpha 1-adrenoceptors were blocked with prazosin (3 microM) and P2-purinoceptors were desensitized with alpha,beta-methylene ATP, the amplitude of contractile responses was reduced by 82-100%. 5. The P2x-purinoceptor antagonists, arylazido amino propyl adenosine triphosphate (ANAPP3) and 5. The P2x-purinoceptor antagonists, arylazido amino propyl adenosine triphosphate (ANAPP3) and suramin, affected LCN-evoked contractile responses. ANAPP3 (50-100 microM) caused a concentration-dependent reduction in the amplitude of contractile response. Suramin (100 microM) caused a small reduction in amplitude of contractile responses but potentiated their amplitude at a concentration of 500 microM. 6. ANAPP3 (100 microM) irreversibly inhibited contractions to alpha,beta-methylene ATP or ATP. Suramin (100-500 microM) inhibited contractions to alpha,beta-methylene ATP (0.5-1 microM) or low concentrations of ATP (10-50 microM) but potentiated contractions at higher concentrations. ANAPP3 (100 microM) and suramin (100, 500 microM) had no effect on contractile responses to noradrenaline. 7. Clonidine (0.05-1 microM), a selective alpha 2-adrenoceptor agonist, caused a concentration-dependent reduction in amplitude of LCN-evoked contractile responses, at 10 Hz, while yohimbine (0.1-1 microM), a selective alpha 2-adrenoceptor antagonist, increased them. At 1 microM, both compounds affected LCN-evoked contractions at all frequencies. This suggests that prejunctional alpha 2-receptors are involved in autoinhibition at sympathetic terminals. 8. In summary, LCN-evoked contractile responses involve the corelease of noradrenaline and ATP or a related purine nucleotide from sympathetic fibres. It is likely that the neurogenic responses are mediated through excitatory postjunctional alpha 1-adrenoceptors, excitatory suramin-sensitive and suramin-insensitiveP2X-purinoceptors and inhibitory beta-adrenoceptors. Also, autoinhibitory prejunctional alpha2-adrenoceptors regulate the LCN excitatory pathway to cat colon circular muscle.

Guanosine 3',5'-cyclic monophosphate regulates calcium channels in neurones of rabbit vesical pelvic ganglia.

1. The effects of dibutyryl guanosine 3',5'-cyclic monophosphate (db-cyclic GMP) were studied in vitro on calcium channels of neurones in rabbit vesical parasympathetic ganglia, using intracellular and single-electrode voltage-clamp recordings. 2. Db-cyclic GMP (100 microM) caused membrane depolarization associated with a decrease in membrane input resistance and an after-hyperpolarization associated with an increase in membrane input resistance. 3. Db-cyclic GMP (0.01-1 mM) caused a concentration-dependent, transient inward current followed by a long-lasting outward current. Membrane conductance was increased and decreased during the inward and outward currents, respectively. 4. The db-cyclic GMP-induced inward current was depressed in nominally calcium-free solutions, by cobalt (1 mM) and nicardipine (10 microM). The mean reversal potentials of the inward current were +42 and -20 mV in the presence and absence of calcium in the external solution, respectively. 5. The db-cyclic GMP-induced inward current was not altered by lowering the external sodium concentration, raising external potassium concentration or by intracellular injection of caesium. 6. A calcium-insensitive component of the db-cyclic GMP-induced current was increased by lowering the external chloride concentration and blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid, a chloride channel blocker. 7. Voltage-dependent, high-threshold calcium currents were depressed during the db-cyclic GMP-induced inward current and facilitated during the outward current. 8. Cyclic GMP was less potent than db-cyclic GMP in causing both inward and outward currents or modulation of calcium currents. GTP, GDP, GMP, guanosine, 8-bromoadenosine 3',5'-cyclic monophosphate and forskolin did not alter the holding current or voltage-dependent calcium currents. 9. It is concluded that intracellular cyclic GMP causes not only activation of resting calcium and chloride channels but also a transient depression followed by long-lasting facilitation of voltage-dependent calcium currents in neurones of vesical parasympathetic ganglia.

Endothelin causes prolonged inhibition of nicotinic transmission in feline colonic parasympathetic ganglia.

The action of endothelin (0.03-1 microM) on neurons in colonic parasympathetic ganglia of cats was studied in vitro, using intracellular microelectrode recording techniques. Electrical stimulation of the pelvic nerve evoked excitatory postsynaptic potentials (EPSPs) and orthodromic action potentials that were reversibly blocked by (+)-tubocurarine, hexamethonium, or external solutions containing nominal zero calcium and elevated magnesium. Endothelin blocked orthodromic action potentials and caused a concentration-dependent prolonged reversible depression of fast EPSPs. Endothelin had minimal effects on nicotinic depolarizations evoked by pressure application of acetylcholine. Endothelin also caused membrane depolarization (2-12 mV) followed by membrane hyperpolarization (1-8 mV). The depolarization and hyperpolarization were associated with a decrease and increase in membrane input resistance, respectively. The actions of endothelin were not altered by superfusion of the ganglia with external solutions containing atropine (300 nM), yohimbine (300 nM), naloxone (1 microM), or substance P (3 microM). We conclude that endothelin modulates synaptic transmission by slow membrane depolarization, membrane hyperpolarization, and prolonged depression of fast EPSPs. We suggest that the blockade of orthodromic action potentials and the depression of fast EPSPs is primarily due to inhibition of release of acetylcholine from presynaptic terminals.

Physiological, morphological, and histochemical properties of cat external anal sphincter.

The contractile properties, morphology, and the distribution of striated muscle fiber types of the external and sphincter (EAS) were determined using axial force measurements, fiber size cross-sectional area measurements, and histochemistry. Electrical stimulation of motor axons in pudendal nerve at supramaximal intensities (10 V, 0.05 ms duration) elicited twitch contractions of EAS. The time to peak force after a single pulse ranged from 37 to 42 ms. The time for relaxation to half-maximal twitch force ranged from 20 to 29 ms. Repetitive stimulation of motor axons (0.1-3.0 Hz) produced potentiation and fatigue of single twitch contractile force, suggesting that the EAS of the cat is comprised predominantly of fast-twitch muscle fibers. Confirmation of skeletal muscle fiber types was determined by histochemistry. Frozen serial cross sections of EAS were incubated to demonstrate succinic dehydrogenase (SDH) and myosin adenosine triphosphatase after alkaline preincubation (pH 10.4). Based on these reactions, muscle fibers were classified as fast glycolytic (FG) (high ATPase, low SDH), fast oxidative-glycolytic (FOG) (high ATPase, high SDH), and slow oxidative (SO) (low ATPase, high SDH). The mean percentage +/- SE of each histochemical type was the following: FG, 73.5 +/- 3.9; FOG, 22.8 +/- 3.7; and SO, 3.7 +/- 0.6. These results indicate that the predominant fiber type for the EAS is FG. The EAS of the cat is considered a nominally fast-twitch muscle.